The identification of the multiple primary targets of a drug or drug candidate is a problem of great importance in the process of drug discovery. In particular, one of the major difficulties in drug discovery is the identification of compounds that have selective actions on specific primary targets.
Two approaches presently dominate the search for new drugs. The first begins with a screen for compounds that have a desired effect on a cell (e.g., induction of apoptosis), or organism (e.g., inhibition of angiogenesis) as measured in a specific biological assay. Compounds with the desired activity may then be modified to increase potency, stability, or other properties, and the modified compounds retested in the assay. Thus, a compound that acts as an inhibitor of angiogenesis when tested in a mouse tumor model may be identified, and structurally related compounds synthesized and tested in the same assay. A critical limitation of this approach is that, often, the mechanisms of action, such as the molecular target(s) and cellular pathway(s) affected by the compound, are unknown, and cannot be determined by the screen. Further, the addition may provide little information about the specificity, either in terms of target or pathways, of the drug's effect. In contrast, the second approach to drug screening involves testing numerous compounds for a specific effect on a known molecular target, typically a cloned gene sequence of an isolated enzyme or protein. For example, high-throughput assays can be developed in which numerous compounds can be tested for the ability to change the level of transcription from a specific promoter or the binding of identified proteins.
The use of high-throughput screens is a powerful methodology for identifying drug candidates, however, it has limitations. In particular, the assay provides little or no information about the effects of a compound at the cellular or organismal level. In order to develop lead compounds into successful drugs, it is necessary not only to find compounds which are able to bind well to the primary target which is being screened, but also to insure that the compounds are not simultaneously interacting with other targets within the cell. These effects must be tested by using the drug in a series of cell biologic and whole animal studies to determine toxicity of side effects in vivo. In fact, analysis of the specificity and toxicity studies of candidate drugs can consume a significant fraction of the drug development process (see, e.g., Oliff et al., 1997, "Molecular Targets for Drug Development," in DeVita et al., Cancer: Principles & Practice of Oncology, 5th Ed., Lippincott-Raven Publishers, Philadelphia, Pa.).
Several gene expression assays are now becoming practicable for quantitating the drug effect on a large fraction of the genes and proteins in a cell culture (see, e.g., Schena et al., 1995, Science 270:467-470; Lockhart et al., 1996, Nature Biotechnology 14:1675-1680; Blanchard et al., 1996, Nature Biotechnology 14:1649; Ashby et al., U.S. Pat. No. 5,569,588, issued Oct. 29, 1996). Raw data from these gene expression assays are often difficult to coherently interpret. Such measurement technologies typically return numerous genes with altered expression in response to a drug, typically 50-100, possibly up to 1,000 or as few as 10. In the typical case, without more analysis, it is not possible to discern cause and effect from such data alone. The fact that one or a few genes among many has an altered expression in a pair of related biological states yields little or no insight into what caused this change and what the effects of this change are. These data in themselves do not inform an investigator about the pathways affected or primary targets of a drug. They do not indicate which effects result from affects on one particular primary target (e.g., the target screened in a high-throughput assay) versus which effects are the result of other primary targets of the drug.
Knowledge of all the primary targets is necessary in understanding efficacy, side-effects, toxicities, possible failures of efficacy, activation of metabolic responses, etc. Further, the identification of all primary targets of a drug can lead to discovery of alternate primary targets suitable to achieve the original therapeutic response. However, without effective methods of analysis, one is left to ad hoc further experimentation to interpret such gene expression results in terms of biological pathways and mechanisms. Systematic procedures for guiding the interpretation of such data and or such experimentation are needed.
Thus, there is a need for improved (e.g., faster and less expensive) systems and methods to identify multiple primary targets in a cell, based on effective interpretation of such data as gene expression data.
Discussion or citation of a reference herein shall not be construed as an admission that such reference is prior art to the present invention.